Ellagitannins are compounds that belong to the class of polyphenols called hydrolyzable tannins, and are complex derivatives of ellagic acid. Recently, it has been suggested that ellagitannins (in addition to the ellagic acid produced after its hydrolysis and the urolitin—which is derived from this molecule due to the action of the intestinal flora) present a series of biological activities that are beneficial to human health. Specifically, among others, these compounds: (1) have a high potential for the treatment of hyperglycemia and hypertension associated to type 2 diabetes (Pinto Mda, S. et al, Journal of Medicinal Food, 2010, 13 (5), 1027-1035), (2) could be used in the treatment of malaria (Dell′Agli, M. et al, Malaria Journal, 2010, 9, 208), (3) have the potential to be used in the prevention of breast cancer (Adams, L. S. et al, Cancer Prevention Research, 2010, 3, 108) and (4) could reduce the risk of developing cancer of the colon (Kasimsetty, S. G. et al, Journal of Agricultural and Food Chemistry, 2010, 58 (4), 2180-2187 and Sharma, M. et al, Journal of Agricultural and Food Chemistry, 2010, 58 (7), 3965-3969). Other studies of plant extracts containing ellagitannins demonstrated their antioxidant and antimicrobial properties as well as their cytotoxic activity against human cancer cells (Barrajón-Catalan, E. et al, Food and Chemical Toxicology, 2010, 48 (8-9), 2273-2282).
In recent years, the pomegranate (Punica granatum L.) has been studied extensively because of the therapeutic applications that have been reported for this fruit since ancient times, and it has been demonstrated recently that this fruit acts as an antioxidant, anti-diabetic and hypolipidemic agent. It has also been observed that this fruit presents antibacterial, anti-inflammatory, antiviral and anti-carcinogenic activities. These benefits have been attributed to the phytochemicals contained in the fruit, which are primarily polyphenols, that, in turn, mainly include hydrolyzable tannins, of which ellagitannins are the most important (Viuda-Martos, M. et al, Comprehensive Reviews in Food Science and Food Safety, 2010, 9 (6), 635-654). The vast potential for the therapeutic use of pomegranate has resulted in that a large part of the scientific literature available on ellagitannins refers to those obtained from this fruit. However, it was recently discovered that the blackberry (the fruit produced by some of several species of the Rubus genus, of the Rosaceae family), specifically Rubus adentrichos Schltdl., could very well have a high ellagitannin content than that of the pomegranate (Acosta-Montoya, O. et al, Food Chemistry, 2010, 119(4), 1497-1501). The high concentrations of these molecules in blackberries at different stages of ripening, make them a raw material of potential interest in the production of extracts that are rich ellagitannins.
The blackberries used in the study mentioned above can be used at the three stages of ripening presented (FIG. 1). The chromatogram segments (high performance liquid chromatography coupled to a diode array detector or HPLC-DAD) at 280 nm of the aqueous—ketone blackberry extract for this study are shown on FIG. 2. As it can be observed, compounds A1, A2 and E1, E2 show spectral features in the ultraviolet visible (UV-visible) of anthocyanins (A1, A2) and ellagitannins (E1, E2). These molecules were identified using liquid chromatography coupled with mass spectrometry (HPLC-MS) and UV visible data from a previous study (Mertz, C. et al, Journal of Agricultural and Food Chemistry, 2007, 55 (21), 8616-8624). Peaks A1 and A2 were tentatively identified as cyanidin glucoside and cyanidin malonyl-glucoside, while peaks E1 and E2 were identified as lambertianin C and sanguin H6. The mentioned study further shown that blackberries of the Rubus glaucus species are also an important source of ellagitannins. Other studies have shown the variety of these compounds that are found in the Apache blackberry (Hager, T. J. et al, Journal of Agricultural and Food Chemistry, 2008, 56 (3), 661-669).
Methods for obtaining extracts from sources that are rich in ellagitannins (purified to a greater or lesser extent) have been extensively investigated, particularly with regard to the use of pomegranates as a source for these compounds. Several scientific publications, primarily patent applications, have been published on this subject.
Document US 2006/0269629 discloses a method for producing an extract containing phytochemicals from pomegranate, by creating a mixture of solids from fruits (pericarp, seeds, etc.) with water, adding enzymatic preparations to degrade the solids (at least partially), increasing the temperature of the mixture and, ultimately, removing the residual insoluble solids. Document US 2006/0211635, in turn, presents a method for the purification of ellagitannins found in pomegranate, by carrying out extraction and purification stages with the implementation of extractions using solvents (such as methanol) and chromatography with solid polymeric adsorbents. In the same manner, document WO 2005/097106 describes a similar technique, based on extraction and purification using a solid polymeric adsorbent.
Document US 2006/0251753 presents a method for the production of an extract containing ellagitannins derived from pomegranate plant materials. To obtain this product, consecutive extractions are carried out with solvents such as ethanol and acetic ether, with the final concentration carried out under vacuum.
Document EP 1967079 discloses a method for the preparation of pomegranate extracts using whole fruits, which are broken up and dispersed in water. After this, the pH is adjusted and clarification through filtration or centrifugation is carried out, adsorption occurs in a non-ionic resin and elution is produced with an aqueous solution. Lastly, it is concentrated by reverse osmosis or nanofiltration and dehydrated by spray drying, vacuum evaporation or lyophilization.
The examples described above have several drawbacks. Some of these processes require the use of solvents for the extraction of the molecules of interest or to carry out desorption of said molecules from the resins (when using solid polymeric adsorbents). Therefore, the final product obtained could contain solvent residues, resulting in an extract that cannot be qualified as “natural”. Other processes described may include the excessive addition of water for the extraction of the compounds of interest from the solids of fruits, which significantly affects the efficiency of the process due to an essential concentration stage would be required, increasing the energy costs. Other techniques require that concentration be carried out under vacuum, which could cause thermal damage to the molecules of interest, thus affecting the functionality of the finished product.
To date, no process has been disclosed that allows for the separation, purification and concentration of ellagitannin, without the use of solvents or high temperatures. In particular, no process has been described that allows separating the polyphenolic compounds of interest (ellagitannins) by using the interactions between their physic-chemical properties and the membrane material, in addition to consider its molar mass.